Le VIH-1 est un rétrovirus contenant deux copies de son génome ARN simple brin positif. Dans la cellule, son ARN est rétrotranscrit en ADN puis cet ADN est intégré dans le génome cellulaire. L'ADN viral est ensuite transcrit en ARN dont la moitié évite l'épissage. Dans le cytoplasme, cet ARN possède une double fonctionnalité : il sert d'ARNm pour la synthèse des protéines Gag et Gag-Pol et d'ARN génomique (ARNg) qui est encapsidé sous forme de dimère. Malgré sa faible abondance dans la cellule, l'ARNg est préférentiellement encapsidé. Cependant, il a été montré par nous et d'autres que d'autres ARN non-génomiques peuvent également être spécifiquement encapsidés, tels que des ARN viraux épissés et certains ARN cellulaires, bousculant ainsi le dogme d'une spécificité unique pour l'ARNg. Nous avons montré que le VIH-1 contrôlerait l'incorporation des ARN viraux, épissés et non épissés, par un mécanisme de compétition, impliquant des facteurs communs. En revanche, les ARN cellulaires 7SL et U6 semblent encapsidés par des mécanismes indépendants. Afin d'identifier les déterminants qui régissent la spécificité d'encapsidation, nous avons testé le rôle de la nucléocapside (NC) qui est fortement impliquée dans l'encapsidation. Nous montrons ici que de façon tou t à fait inattendue, des mutations de NC conduisent à l'encapsidation d'ADN et augmentent également l'encapsidation d'ARN viraux épissés. Nous avons ensuite cartographié les déterminants de la région 5' UTR de l'ARNg et déterminé que la tige-boucle polyA et la région PBS sont impliquées dans l'encapsidation des ARN viraux épissés. Ce travail apporte une meilleure compréhension de la spécificité d'encapsidation, primordiale pour la mise au point de thérapies géniques utilisant des vecteurs lentiviraux. / HIV-1 is a retrovirus containing two copies of its single stranded positive RNA genome. In the cell, its RNA is reverse transcribed into DNA and this DNA is integrated into the cellular genome. The viral DNA is then transcribed into RNA. One moiety of this RNA pool escapes splicing. Once in the cytoplasm, this RNA has a double function: it serves as mRNA for synthesis of Gag and Gag-Pol proteins and as genomic RNA (gRNA) that is packaged as a dimer. Despite its low abundance in the cell, the gRNA is preferentially encapsidated into virions. However, it has been shown by us and others that other non-genomic RNA can be specifically packaged, such as spliced viral RNA and some cellular RNA, thus shaking up the dogma of a unique specificity for the gRNA. We have shown that HIV-1 might control the incorporation of the spliced and unspliced RNA by a mechanism of competition, involving common factors. In contrast, cellular RNA 7SL and U6 seem encapsid ated through independent mechanisms. To identify the determinants that govern the specificity of encapsidation, we tested the role of the nucleocapsid (NC) which is strongly involved in the packaging. Here we show that unexpectedly, mutations of NC lead to the encapsidation of DNA and also increase the encapsidation of spliced viral RNA. We then mapped the determinants in the 5' UTR of the gRNA and determined that the polyA stem-loop and the PBS region are involved in the encapsidation of the spliced viral RNA. This work provides a better understanding of the specificity of encapsidation that is crucial for the development of gene therapy using lentiviral vectors.

Gold compounds have been used in medicine dating back as early as 2500 BC. Over the years gold(I) and gold(III) compounds have been used and designed to target rheumatoid arthritis, cancer, and viral diseases. New drug targets have been found for gold compounds that give insight into their mechanisms of action. Here we focus on the synthesis of Au(III) compounds designed to selectively target zinc finger (ZF) proteins. ZF proteins exhibit a variety of functions, including transcription, DNA repair, and apoptosis. Displacement of the central zinc ion, along with mutation of coordinated amino acids can result in a loss of biological function. Synthesis of complexes that selectively target zinc finger proteins, in turn inhibiting DNA/ZF interactions and therefore resulting in loss of protein function, is of great interest. Of particular interest here is the Cys3His (Cys = cysteine, His = histidine) HIV nucleocapsid zinc finger protein, NCp7. NCp7 is involved in multiple steps of the HIV life cycle, thus making it a desirable drug target. Previous studies from our group show platinated nucleobases such as [Pt(dien)(9-EtG)]2+ (dien = diethylenetriamine; 9-EtG = 9-ethylguanine) to stack effectively in a non-covalent manner with tryptophan of the C-terminal finger of HIV Nucleocapsid, NCp7(F2), a key residue involved in nucleic acid recognition. Due to the isoelectronic and isostructural relationship of Au(III) to Pt(II), we have expanded this system to Au(III)-(nucleobase/N-heterocycle) compounds. Novel Au(III)(dien)(N-heterocycle) compounds, including the first Au(III)N3(N-purine) examples, were synthesized. As previously reported for [AuCl(dien)]Cl2, these compounds exhibit pH dependency of the 1H NMR chemical shifts of the dien ligand. The acidity of the dien ligand is affected by the nature of the fourth ligand as a leaving group. The presence of an inert nitrogen donor, compared to that of the more labile Cl-, as the leaving group stabilizes the Au(III) metal center towards reduction, resulting in significant enhancement of π−π stacking interactions with tryptophan relative to platinum(II) and palladium(II) compounds. The presence of a more inert N-donor as the leaving group slows down the reaction with the sulfur-containing amino acid N-Acetylmethionine (N-AcMet); essentially no reaction was observed for the Au(III)-N-heterocycle compounds. All compounds react readily with N-Acetylcysteine (N-AcCys), however lack of N-heterocycle ligand dissociation indicates, to our knowledge, the first long-lived N-heterocycle-Au-S species in solution. Electrospray ionization mass spectrometry (ESI-MS) studies with NCp7(F2) indicate [Au(dien)(DMAP)]3+ (DMAP = 4-dimethylaminopyridine) to be the least reactive of the Au(III) compounds studied, showing the presence of intact NCp7(F2) zinc finger at initial reaction times. Reactivity of the Au-compounds was compared with that of Sp1(F3), a Cys2His2 ZF; in contrast, no intact ZF was observed for any of the compounds studied, suggesting the mode of action of these compounds is dependent on the nature of the zinc binding core. ESI-MS studies were expanded to that of the full HIV NCp7 zinc finger. [Au(dien)(9-EtG)]3+ reacts quickly with NCp7, resulting in immediate zinc ejection and replacement with up to three gold ions. Unlike with [Au(dien)(DMAP)]3+, no intact NCp7 was observed. Addition of [Au(dien)(9-EtG)]3+ to preformed NC-SL2 complex results in release of free RNA; based on EMSA (electrophoretic mobility shift assay) studies, [Au(dien)(9-EtG)]3+ disrupts the NCp7-RNA complex with an IC50 of ~450 µM. It is possible that this HIV nucleocapsid-nucleic acid antagonism may result in a loss of viral activity.

O vírus Oropouche (OROV) é um arbovírus que ocorre na região amazônica causando surtos de doenças febris agudas e que, ocasionalmente, podem ser associados a meningoencefalite. Aproximadamente 500.000 casos de Oropouche teriam ocorrido no Brasil. Entretanto, não existe vacina contra o OROV. O objetivo deste trabalho foi desenvolver um modelo animal de infecção por OROV para estudar a patogênese da doença e um modelo para testar candidatas vacinais. Protótipo vacinal utilizando a proteína recombinante do nucleocapsídeo (N) de OROV (NrOROV), que é o principal antígeno viral, foi usado como potencial candidato para vacina. Neste estudo utilizou-se um modelo animal em camundongos Balb/c de 12 semanas de idade, inoculados intracerebralmente com 8x105 PFU de OROV, capaz de induzir 100% de letalidade após o terceiro dia da infecção. Altos títulos virais foram encontrados no cérebro e na medula espinhal dos animais. Surpreendentemente, 12 e 24 horas pós-infecção foi possível detectar vírus no fígado e baço (3 Log10 PFU/g) dos camundongos. Com este modelo foram testados os candidatos vacinais. Grupos de camundongos foram imunizados 3 vezes com OROV, OROV e FCA, NrOROV, NrOROV e FCA, NrOROV, Poli I:C e Montanide ISA 720. Após 3 imunizações, os animais foram desafiados com 10 LD50 de OROV e observados por 20 dias. Os animais imunizados com NrOROV e adjuvantes, não foram capazes de produzir anticorpos neutralizantes e adquirir imunidade protetora contra OROV enquanto que os imunizados com OROV apresentaram altos níveis de anticorpos neutralizantes e completa proteção in vivo. Ainda, os anticorpos produzidos pelos animais imunizados permitiram estudar o ciclo de replicação celular do OROV utilizando imunofluorescência. / Oropouche (OROV) is an arbovirus that occurs in the South American, Amazon region, producing outbreaks of acute febrile illness occasionally associated to meningoencephalitis. Approximately 500,000 cases of Oropouche have been reported in Brazil in the last 60 years. However, there is no available vaccine for OROV. We show here the development of an animal model of OROV suitable for studies on pathogenesis and vaccine testing. A vaccine prototype based on recombinant OROV nucleocapsid protein (NrOROV), an important viral antigen, was evaluated in the animal model. Initialy, we observed that all 12-week-old Balb/c mice inoculated intracerebrally with 8x105 PFU died after the third day of infection. Surprisingly, OROV genome was detectable in the liver as early as 12 hours post infection (pi) and in the spleen at 24 hours pi at 3 log10 PFU/g. Besides, high viral titers were found in brain and spinal cord. To test the NrOROV as a vaccine candidate, animals divided in 5 groups were immunized subcutaneously 3 times, two weeks apart with either OROV, OROV and Freud complete Adjuvant (FCA), NrOROV, NrOROV and FCA, NrOROV and Poly I:C and Montanide ISA 720. The experiment also included a group of naïve animals. After the third immunization, the animals were challenged with 10LD50 by intracerebral route and followed for 20 days. The animals immunized with NrOROV and adjuvants developed specific antibodies that were not able to neutralize the virus or confer protective immunity against OROV. Nevertheless, mice immunized with OROV showed high levels of neutralizing and protective antibodies. Despite the discouraging results with NrOROV as a vaccine, the mouse model is suitable to study pathogenesis, and to test other vaccines for OROV.

O vírus Oropouche (OROV) é um arbovírus que ocorre na região amazônica causando surtos de doenças febris agudas e que, ocasionalmente, podem ser associados a meningoencefalite. Aproximadamente 500.000 casos de Oropouche teriam ocorrido no Brasil. Entretanto, não existe vacina contra o OROV. O objetivo deste trabalho foi desenvolver um modelo animal de infecção por OROV para estudar a patogênese da doença e um modelo para testar candidatas vacinais. Protótipo vacinal utilizando a proteína recombinante do nucleocapsídeo (N) de OROV (NrOROV), que é o principal antígeno viral, foi usado como potencial candidato para vacina. Neste estudo utilizou-se um modelo animal em camundongos Balb/c de 12 semanas de idade, inoculados intracerebralmente com 8x105 PFU de OROV, capaz de induzir 100% de letalidade após o terceiro dia da infecção. Altos títulos virais foram encontrados no cérebro e na medula espinhal dos animais. Surpreendentemente, 12 e 24 horas pós-infecção foi possível detectar vírus no fígado e baço (3 Log10 PFU/g) dos camundongos. Com este modelo foram testados os candidatos vacinais. Grupos de camundongos foram imunizados 3 vezes com OROV, OROV e FCA, NrOROV, NrOROV e FCA, NrOROV, Poli I:C e Montanide ISA 720. Após 3 imunizações, os animais foram desafiados com 10 LD50 de OROV e observados por 20 dias. Os animais imunizados com NrOROV e adjuvantes, não foram capazes de produzir anticorpos neutralizantes e adquirir imunidade protetora contra OROV enquanto que os imunizados com OROV apresentaram altos níveis de anticorpos neutralizantes e completa proteção in vivo. Ainda, os anticorpos produzidos pelos animais imunizados permitiram estudar o ciclo de replicação celular do OROV utilizando imunofluorescência. / Oropouche (OROV) is an arbovirus that occurs in the South American, Amazon region, producing outbreaks of acute febrile illness occasionally associated to meningoencephalitis. Approximately 500,000 cases of Oropouche have been reported in Brazil in the last 60 years. However, there is no available vaccine for OROV. We show here the development of an animal model of OROV suitable for studies on pathogenesis and vaccine testing. A vaccine prototype based on recombinant OROV nucleocapsid protein (NrOROV), an important viral antigen, was evaluated in the animal model. Initialy, we observed that all 12-week-old Balb/c mice inoculated intracerebrally with 8x105 PFU died after the third day of infection. Surprisingly, OROV genome was detectable in the liver as early as 12 hours post infection (pi) and in the spleen at 24 hours pi at 3 log10 PFU/g. Besides, high viral titers were found in brain and spinal cord. To test the NrOROV as a vaccine candidate, animals divided in 5 groups were immunized subcutaneously 3 times, two weeks apart with either OROV, OROV and Freud complete Adjuvant (FCA), NrOROV, NrOROV and FCA, NrOROV and Poly I:C and Montanide ISA 720. The experiment also included a group of naïve animals. After the third immunization, the animals were challenged with 10LD50 by intracerebral route and followed for 20 days. The animals immunized with NrOROV and adjuvants developed specific antibodies that were not able to neutralize the virus or confer protective immunity against OROV. Nevertheless, mice immunized with OROV showed high levels of neutralizing and protective antibodies. Despite the discouraging results with NrOROV as a vaccine, the mouse model is suitable to study pathogenesis, and to test other vaccines for OROV.

by Chan Wai Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 170-185). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 摘要 --- p.v / Table of Content --- p.vii / Abbreviations --- p.xii / for Nucleotides --- p.xii / for Amino acids --- p.xii / for Standard genetic codes --- p.xiii / for Units --- p.xiii / for Prefixes of units --- p.xiv / for Terms commonly used in the report --- p.xiv / List of Figures --- p.xvii / List of Tables --- p.xxiii / Chapter Chapter I --- Introduction --- p.1 / Chapter 1.1 --- Epidemiology of the Severe Acute Respiratory Syndrome --- p.1 / Chapter 1.2 --- The SARS Coronavirus --- p.3 / Chapter 1.3 --- Cell Biology of Coronavirus Infection and Replication and the Role of Nucleocapsid Protein --- p.9 / Chapter 1.4 --- Recent Advances in the SARS-CoV Nucleocapsid Protein --- p.16 / Chapter 1.5 --- The Sumoylation System --- p.24 / Chapter 1.6 --- Objectives of the Present Study --- p.28 / Chapter Chapter II --- SARS-CoV N protein and Fragment Purification --- p.29 / Chapter 2.1 --- INTRODUCTION --- p.29 / Chapter 2.2 --- METHODOLOGY --- p.31 / Materials --- p.31 / Methods --- p.39 / Chapter 2.2.1 --- Construction of the pMAL-c2P vector --- p.39 / Chapter 2.2.2 --- Sub-cloning of the N protein into expression vectors --- p.42 / Chapter 2.2.2.1 --- Design of primers for the cloning of N protein --- p.43 / Chapter 2.2.2.2 --- DNA amplification using Polymerase Chain Reaction (PCR) --- p.44 / Chapter 2.2.2.3 --- DNA extraction from agarose gel --- p.45 / Chapter 2.2.2.4 --- Restriction digestion of purified PCR product and vectors --- p.46 / Chapter 2.2.2.5 --- Ligation of N protein into expression vectors --- p.47 / Chapter 2.2.2.6 --- Preparation of competent cells --- p.48 / Chapter 2.2.2.7 --- Transformation of plasmids into competent Escherichia coli --- p.49 / Chapter 2.2.2.8 --- Preparation of plasmid DNA --- p.49 / Chapter 2.2.2.8.1 --- Mini-preparation of plasmid DNA --- p.49 / Chapter 2.2.2.8.2 --- Midi-preparation of plasmid DNA --- p.51 / Chapter 2.2.3 --- Expression of tagged and untagged N protein --- p.53 / Chapter 2.2.3.1 --- Preparation of E. coli competent cells for protein expression --- p.53 / Chapter 2.2.3.2 --- Expression of N protein --- p.53 / Chapter 2.2.3.3 --- Solubility tests on the fusion proteins expressed --- p.54 / Chapter 2.2.4 --- Purification of N protein Chromatographic methods --- p.55 / Chapter 2.2.4.1 --- Affinity chromatography --- p.55 / Chapter 2.2.4.1.1 --- Ni-NTA affinity chromatography --- p.55 / Chapter 2.2.4.1.2 --- Glutathione affinity chromatography --- p.56 / Chapter 2.2.4.1.3 --- Amylose affinity chromatography --- p.56 / Chapter 2.2.4.2 --- Ion exchange chromatography --- p.57 / Chapter 2.2.4.2.1 --- Cation exchange chromatography --- p.57 / Chapter 2.2.4.2.2 --- Anion exchange chromatography --- p.58 / Chapter 2.2.4.3 --- Heparin affinity chromatography --- p.58 / Chapter 2.2.4.4 --- Size exclusion chromatography Purification strategies --- p.60 / Chapter 2.2.4.5 --- Purification of His6-tagged N proteins --- p.60 / Chapter 2.2.4.6 --- Purification of MBP-tagged N proteins --- p.60 / Chapter 2.2.4.7 --- Purification of GST-tagged N proteins --- p.61 / Chapter 2.2.4.8 --- Purification of untagged N proteins --- p.61 / Chapter 2.2.5 --- Trypsin digestion assay for the design of stable fragment --- p.64 / Chapter 2.2.6 --- Partial purification of the N protein amino acid residue 214-422 fragment --- p.65 / Chapter 2.2.7 --- Sumoylation of the SARS-CoV N protein --- p.67 / Chapter 2.2.7.1 --- In vitro sumoylation assay --- p.67 / Chapter 2.2.7.2 --- Sample preparation for mass spectrometric analysis --- p.68 / Chapter 2.3 --- RESULTS --- p.70 / Chapter 2.3.1 --- Construction of the vector pMAL-c2P --- p.70 / Chapter 2.3.2 --- "Construction of recombinant N protein-pAC28m, N-protein- pGEX-6P-l,N protein-pMAL-c2E and N protein-pMAL-c2P plasmids" --- p.72 / Chapter 2.3.3 --- Optimization of expression conditions --- p.79 / Chapter 2.3.4 --- Screening of purification strategies --- p.82 / Chapter 2.3.4.1 --- Purification of His6-N protein --- p.82 / Chapter 2.3.4.2 --- Purification of MBP-N protein --- p.84 / Chapter 2.3.4.3 --- Purification of GST-N protein --- p.85 / Chapter 2.3.4.4 --- Purification of untagged N protein --- p.87 / Chapter 2.3.5 --- Limited trypsinolysis for the determination of discrete structural unit --- p.91 / Chapter 2.3.6 --- Partial purification of the N protein 214-422 fragment --- p.94 / Chapter 2.3.7 --- Sumoylation of N protein --- p.97 / Chapter 2.2.7.1 --- Sumoylation site prediction --- p.97 / Chapter 2.2.7.2 --- In vitro sumoylation assay --- p.99 / Chapter 2.2.7.3 --- Mass spectrometric identification of sumoylated SARS-CoV N protein --- p.103 / Chapter 2.4 --- DISCUSSION --- p.109 / Chapter Chapter III --- Characterization of the Nucleic Acid Binding Ability of N protein --- p.119 / Chapter 3.1 --- INTRODUCTION --- p.119 / Chapter 3.2 --- METHODOLOGY --- p.120 / Materials --- p.120 / Methods --- p.124 / Chapter 3.2.1 --- Spectrophotometric Measurement of ratio OD260/ OD280 --- p.124 / Chapter 3.2.2 --- Native gel electrophoresis --- p.124 / Chapter 3.2.3 --- Quantitative determination of nucleic acids content --- p.125 / Chapter 3.2.3.1 --- Dische assay - quantitative determination of DNA content --- p.125 / Chapter 3.2.3.2 --- Orcinol assay - quantitative determination of RNA content --- p.126 / Chapter 3.2.4 --- RNase digestion of the N protein-bound RNA --- p.128 / Chapter 3.2.5 --- Isolation of RNA from purified GST-N proteins --- p.128 / Chapter 3.2.6 --- In vitro transcription of SARS-CoV genomic RNA fragment --- p.129 / Chapter 3.2.7 --- Vero E6 cell line maintenance and total RNA extraction --- p.131 / Chapter 3.2.8 --- Electrophoretic mobility shift assay (EMSA) --- p.131 / Chapter 3.3 --- RESULTS --- p.133 / Chapter 3.3.1 --- Detection of nucleic acids in the purified N proteins byspectrophotometric Measurement of ratio OD260/ OD280 --- p.133 / Chapter 3.3.2 --- Native gel electrophoresis --- p.135 / Chapter 3.3.3 --- Quantitative determination of nucleic acids content in purified GST-N proteins --- p.136 / Chapter 3.3.3.1 --- Dische assay for the determination of DNA --- p.136 / Chapter 3.3.3.2 --- Orcinol assay for the determination of RNA --- p.138 / Chapter 3.3.4 --- RNase digestion treatment --- p.139 / Chapter 3.3.5 --- Extraction of RNA from GST-N proteins --- p.140 / Chapter 3.3.6 --- In vitro transcription of SARS-CoV genomic RNA fragment --- p.142 / Chapter 3.3.7 --- Electrophoretic mobility shift assay (EMSA) --- p.144 / Chapter 3.4 --- DISCUSSION --- p.147 / Chapter Chapter IV --- Discussion --- p.154 / Chapter 4.1 --- "Purity, Aggregation and RNA Binding Property of the SARS-CoV Nucleocapsid Protein" --- p.154 / Chapter 4.2 --- Future perspectives --- p.156 / Chapter 4.2.1 --- Structural study of the SARS-CoV N protein through x-ray crystallography --- p.156 / Chapter 4.2.2 --- Mapping the RNA binding domain in the SARS-CoV N protein --- p.156 / Chapter 4.2.3 --- Determination of aggregation state by lateral turbidimetry analysis --- p.156 / Chapter 4.2.4 --- Exploring protein interacting partners that enhance RNA binding specificity --- p.157 / Appendix --- p.159 / Chapter I. --- Sequence of the SARS-CoV N protein --- p.159 / Chapter II. --- Sequence of the SARS-CoV genome fragment used for RNA binding assay in section 3.37.1 --- p.161 / Chapter III. --- Vector maps --- p.161 / Chapter a) --- Vector map of pACYC177 --- p.161 / Chapter b) --- Vector map and MCS of pET28a --- p.163 / Chapter c) --- Vector map and MCS of pAC28 --- p.164 / Chapter d) --- Vector map and MCS of pGEX-6P-1 / Chapter e) --- Vector map of pMAL-c2X and MCS of pMAL-c2E / Chapter IV. --- Electrophoresis markers --- p.166 / Chapter V. --- SDS-PAGE gel parathion protocol --- p.169 / References --- p.170

The dissertation describes development and characterization of monoclonal antibodies against recombinant yeast-expressed antigens: nucleocapsid (N) proteins of human parainfluenza virus type 3, Menangle virus, hantavirus and rabies virus. The newly developed antibodies were investigated by different immunochemical assays for their specificity, affinity and ability to recognize native viruses in infected cells. It was determined that the antibodies raised against recombinant yeast-expressed viral proteins are suitable to identify virusinfected cells. These data confirmed that recombinant yeast-expressed viral N proteins possess antigenic properties similar to that of native viral nucleocapsids. The monoclonal antibodies were also used to study the antigenic structure of viral N proteins and localize their immunodominant regions. The obtained results may have impact on the development of new immunodiagnostic test systems for the detection of viral infections. The dissertation consists of the introduction, three sections, references, and the list of author’s publications. In the Introductory Chapter, the research topic, the actuality, the aim and tasks, scientific novelty and practical value of the dissertation are discussed. Author’s publications and conference reports are also presented. The first Chapter of the dissertation provides literature overview on the genome organization, structural proteins, pathogenesis and epidemiology of parainfluenza viruses, Menangle virus... [to full text] / Disertacijoje aprašomi monokloniniai antikūnai, sukurti prieš rekombinantinius mielėse susintetintus antigenus: žmogaus paragripo treciojo tipo viruso, Menangle viruso, hantavirusų bei pasiutligės viruso nukleokapsidės (N) baltymus. Sukurtieji antikūnai buvo visapusiškai charakterizuoti įvairiais imunocheminės analizės metodais, įvertintas jų specifiškumas, afiniškumas, sugebėjimas atpažinti natyvius virusus infekuotų ląstelių kultūrose. Buvo nustatyta, kad antikūnai, sukurti prieš rekombinantinius mielėse susintetintus virusų baltymus, tinka virusų nustatymui infekuotose ląstelėse. Šie tyrimai patvirtino, kad rekombinantiniai mielėse susintetinti virusu N baltymai turi panašias antigenines savybes, kaip natyvūs virusų N baltymai, formuojantys nukleokapsides. Sukurtieji monokloniniai antikūnai taip pat buvo panaudoti išsamiems minėtų virusų N baltymų antigeninės struktūros tyrimams bei imunodominuojančių sekų nustatymui. Disertaciniame darbe gauti duomenys svarbūs, kuriant naujas imunodiagnostikos sistemas, skirtas virusų infekcijoms nustatyti. Disertacija sudaro įvadas, trys skyriai, naudotos literatūros sąrašas ir autorės publikacijų sąrašas. Įvadiniame skyriuje aptariama tiriamoji problema, darbo aktualumas, formuluojamas darbo tikslas bei uždaviniai, darbo mokslinis naujumas ir praktinė reikšmė, pristatomos paskelbtos publikacijos ir pranešimai konferencijose. Pirmasis disertacijos skyrius skirtas literatūros apžvalgai: jame apibūdinamos paragripo virusų, Menangle viruso... [toliau žr. visą tekstą]

Thesis (M.S.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains iv, 66, [6] p. : ill. Includes abstract. Includes bibliographical references.

Orientadora: Profa. Dra. Maria Aparecida Sperança / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2016. / A família Bunyaviridae agrupa virus de RNA trissegmentados de cadeia negativa e inclui mais de 350 isolados classificados em cinco gêneros, Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus e Tospovirus. Juntos, esses bunyavirus infectam uma grande variedade de animais e plantas, e determinados vírus têm a capacidade de causar doenças graves em seus respectivos hospedeiros. Depois da febre da Dengue, a doença viral transmitida por artrópodes com maior prevalência no Brasil corresponde à febre do Oropouche (FO), cujo agente etiológico é o arbovírus Oropouche (OROV) da família Bunyarviridae, gênero Orthobunyavírus, grupo sorológico Simbu. Nos últimos anos, OROV tem sido responsável por mais de 500.000 casos humanos em extensas e explosivas epidemias na Região Amazônica e no Planalto Central. Com o intuito de buscar novas ferramentas para o diagnóstico específico e tratamento da febre do Oropouche, este projeto teve por objetivo investigar a forma recombinante da proteína do Nucleocapsídeo (N) por meio de estudos estruturais. A proteína N dos bunyavirus é essencial para o processo de replicação, apresentando mecanismo específico de encapsidação do genoma de RNA. O gene que codifica a proteína N de OROV foi subclonada no vetor de expressão bacteriano pET28a (+), com subseqüente expressão em bactéria E. coli da linhagem BL21 (DE3), em diferentes condições de temperatura. Desenvolveram-se estudos estruturais com a análise de espalhamento dinâmico de luz (DLS), Dicroísmo circular (CD), e de espalhamento de raios X a baixos ângulos (SAXS). Os resultados indicaram que o monômero da proteína N de OROV produzida em bactérias apresenta 28kDa com cauda de histidina, e após purificação em condições nativas, apresentou alta massa molecular com tamanho entre 400-660 kDa, sugerindo a formação de multímero associado inespecificamente à molécula de RNA com tamanho menor de 100 bases. / The Bunyaviridae family groups trissegmented RNA virus of negative strand and includes more than 350 isolates classified into five genders, Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus and Tospovirus. Together, these bunyavirus infect a variety of animals and plants, and certain viruses have the ability to cause severe disease in their hosts. After Dengue fever, the viral disease transmitted by arthropods with a higher prevalence in Brazil corresponds to Oropouche fever (OF), whose etiologic agent is the Oropouche virus (OROV) of Bunyarviridae family, Orthobunyavirus genus, and serologic group Simbu. In recent years, OROV has been responsible for more than 500,000 human cases in extensive and explosive epidemics in the Amazon region and in the Central Highlands. In order to search for new tools for specific diagnosis and treatment of OF, this project aimed to investigate the recombinant form of the nucleocapsid (N) protein by means of structural studies. The N protein of bunyavirus is essential for the replication process, and present a specific mechanism for encapsidation of the virus RNA genome. The gene encoding the OROV N protein was subcloned into the bacterial expression vector pET28a (+) with subsequent expression in E. coli strain BL21 (DE3) under different temperature conditions. Structural studies have been developed with the dynamic light scattering analysis (DLS), circular dichroism (CD), and X-ray scattering at low angles (SAXS). The results indicated that the monomer OROV N protein with six histidine tag, produced in bacteria, has 28kDa, and after purification under native conditions, showed high molecular mass size between 400-660 kDa, suggesting multimer formation associated nonspecifically to RNA molecules with a length less than 100 bases.

La protéine de nucléocapside (NC) du virus de l’immunodéficience humaine (VIH-1) joue de nombreux rôles dans les phases précoce et tardive de l’infection. La NC est une protéine à deux doigts de zinc, chaperonne des acides nucléiques. Nous avons cherché de nouveaux partenaires cellulaires de la NCp7 et identifié une protéine de liaison aux ARNs, Upstream of N-ras (Unr), dont l’interaction avec Gag et NCp7 a été confirmée. L’interaction entre Gag et Unr est dépendante de l’ARN et médiée par le domaine NC. Unr est une ITAF (IRES transacting factor) régulant la traduction médiée par plusieurs IRESs cellulaires et viraux. L’ARN génomique du VIH-1 possède deux IRESs dont un localisé dans la région non traduite en 5’ qui permet aux ARNm viraux de conserver un fort niveau de traduction lorsque la traduction coiffe-dépendante de la cellule est affaiblie par l’arrêt du cycle viral induit par l’infection. En utilisant un système de dual luciférase, nous avons montré qu’Unr est une ITAF dont la surexpression stimule l’IRES VIH-1. Des mutations ponctuelles de cet IRES, dans un motif consensus de liaison à Unr, altèrent à la fois l’activité de l’IRES et sa réponse à Unr suggérant que l’activité IRES dépend fortement de Unr. L’effet d’Unr sur l’IRES est inhibé par la surexpression de NCp7 mais pas par celle de Gag dont l’effet stimulateur sur l’IRES est additif de celui d’Unr suggérant un rôle d’Unr différent dans les phases précoce et tardive de l’infection. Pour finir, le knockdown de l’expression d’Unr entraîne une diminution significative de l’infection par un pseudovirus non réplicatif soulignant l’implication fonctionnelle d’Unr dans la phase précoce. / The Human Immunodeficiency Virus-1 (HIV-1) nucleocapsid protein (NC), as a mature protein (NCp7) or as a domain of the polyprotein Gag, plays several important roles in both the early and late phase of the infection. NC is a nucleic acid chaperone protein with two zinc fingers. We searched for new cellular protein partners of NCp7 and identified the RNA binding protein Unr, Upstream of N-ras, whose interaction with both Gag and NCp7 was confirmed. Unr interaction with Gag is RNA dependent and mediated by its NC domain. Unr is an ITAF (IRES trans-acting factor) regulating the translation driven by several IRESs. The HIV-1 genomic mRNA harbors two IRESs elements: one of them found within the HIV-1 5’-Untranslated region drives HIV-1 mRNA translation when the cap-dependent translation is diminished due to the infection-induced cell cycle arrest. Using a dual luciferase assay, Unr was shown to act as an ITAF, increasing the HIV-1 IRES dependent translation. Point mutations of the HIV-1 IRES in a consensus Unr binding motif were found to alter both the IRES activity and its activation by Unr suggesting a strong dependency of the IRES on Unr. Unr stimulation effect is furthermore counteracted by NCp7, but not by Gag overexpression, which increases the IRES activity in an additive manner to Unr suggesting a differential Unr effect on the early and late phases of the infection. Finally, knockdown of Unr in HeLa cells leads to a decline in infection by a non-replicative lentivector proving its functional implication in the early phase.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Raymond R. Rowland / Severe acute respiratory syndrome (SARS) is caused by an enveloped, positive-stranded RNA virus, the SARS coronavirus (SARS-CoV). Coronaviruses along with the arteriviruses are placed in the order, Nidovirales. Even though nidovirus replication is restricted to the cytoplasm, the nucleocapsid protein (N) of several coronaviruses and arteriviruses, localize to the nucleolus during infection. Confocal microscopy of N protein localization in Vero cells infected with the SARS-CoV or transfected with the SARS-CoV N gene failed to show presence of N in the nucleoplasm or nucleolus. Recombinant N remained cytoplasmic after the addition of leptomycin B (LMB), a drug that inhibits nuclear export. SARS-CoV N possesses a unique lysine-rich domain, located between amino acids 369-389, which possesses several nuclear localization signal (NLS) and nucleolar localization signal (NoLS) motifs. A chimeric protein composed of the 369-389 peptide substituted for the NLS of equine infectious anemia virus (EIAV) Rev protein (ERev) showed no nuclear localization activity. Three negatively charged amino acids, located at positions 372, 377 and 379 in SARS-CoV N were hypothesized to play a role in the loss of nuclear targeting. Substitution of aspartic acid-372 with alanine restored nuclear localization to the chimeric protein. A full-length recombinant SARS-N protein with the alanine-372 substitution localized to the nucleus. Therefore, the presence of an aspartic acid at position 372 is sufficient to retain N in the cytoplasm The mechanistic basis for how aspartic acid-372 interrupts nuclear transport is unknown, but may lie in the electrostatic repulsion with negatively charged amino acids located within the NLS binding pocket of importin-alpha.